Chemokine receptor peptide vaccines for treatment and prevention of diabetes

ABSTRACT

The present invention provides immunogenic oligopeptides derived from the chemokine receptor protein for use in compositions and methods for the treatment, and prevention of inflammatory responses.

BACKGROUND OF THE INVENTION

[0001] The present invention relates to novel compositions and methodsfor inhibiting inflammatory responses associated with autoimmunediseases. In particular, it relates to vaccination with peptides fromthe extracellular regions of chemokine receptor molecules.

[0002] Chemokines constitute a family of small molecular weightcytokines that are produced in inflammation and regulate leukocyterecruitment. These molecules are ligands for seven transmembrane Gprotein linked receptors that induce a signaling cascade costimulationfor T cell activation in addition to participating in transendothelialmigration of leukocytes (Oppenheim et al. Ann. Rev. Immunol. 9:617-648(1991), Premback et al. Nat. Med. 2:1174-1178 (1996)). Two subfamiliesof chemokines, referred to as CC and CXC, have been discovered. CC andCXC chemokines are distinct from each other in their N terminal aminoacid sequence which starts either with cysteine-cysteine orcysteine-X-cysteine where X is typically another L-amino acid. They arealso distinct in their binding pattern to their receptors. For example,the CC chemokines bind to CC receptors and not to CXC receptors and viceversa.

[0003] Different chemokines regulate the trafficking of distinctpopulations of hemopoietic cells by activating specific 7-transmembranereceptors expressed by these cells (Baggiolini et al. Adv. Immunol.55:97-179 (1994); Gerard et al. Curr. Opin. Immunol.6:140-145 (1994)).Recent publications indicate that the Th1 and Th2 subsets of regulatoryT cells are uniquely characterized by the chemokine receptors CXCR3 andCCR3, respectively (Sallusto, et al. J. Exp. Med. 187:875-883 (1998);Bonecchi, et al. J Exp. Med. 187:129-134 (1998); Qin, et al. J. ClinInvest. 101:746-754 (1998)). Several studies have correlated theexpression of three specific chemokines, IP-10, RANTES, and MCP-1,produced by astrocytes in the CNS with the presence of inflammatoryinfiltration within this tissue during the early phase of EAE(Ransohoff, et al. FASEB J. 7:592-600 (1993); Glabinski et al. Am. J.Pathol. 150:617-630 (1995) Godiska, et al. J. Neuroimmunol. 58:167-176(1995); and Eng et al. Neurchem. Res. 21:511-525 (1996)). While allthree chemokines have been shown to be capable of recruiting Tlymphocytes in certain experimental models, IP-10 has been demonstratedto be specific for this lineage of hemopoietic cells (Taub et al. J.Exp. Med. 177:1809-1814 (1993)); Carr, et al. Proc. Natl. Acad. Sci. USA91:3652-3656 (1994); and Farber, J. Leukoc. Biol. 61:246-257 (1997).MBP-immunized rats intrathecaly infused with an antisensephosphorothioate oligonucleotide to crg-2 (the murine homologue of humanIP-10) show reduced disease clinical score of EAE (Wojcik, et al. J.Pharmacol. Exp. Ther. 278:404-410 (1996)). In addition, higherexpression of some of the chemokine receptors such as CXCR3 on IL2activated human T lymphocytes and not on resting T lymphocytes has beendemonstrated (Loetscher et al. J Exp. Med. 184:963-969 (1996)).

[0004] Multiple sclerosis (MS) is a T cell-dependent autoimmune diseasecaused by localized demyelination in the central nervous system (CNS),with only limited therapeutic options available to patients. Extensiveinvestigation has indicated that these autoreactive T lymphocytesfrequently, though not always, express the Th1 phenotype of high levelproduction of IFNg, IL-2 and TNFa, with little to no IL-4, IL-5 andIL-10.

[0005] Current therapeutics for autoimmune diseases, such as MS, involvethe use of antiinflammatory agents or general immunosuppressants. Priorart methods for controlling autoimmune disease fail to provide a simpleself-mediated method for specifically eliminating inflammatory responsesmediated by chemokines associated with the autoimmune responses. Thepresent invention addresses these and other needs.

SUMMARY OF THE INVENTION

[0006] The present invention provides methods of inducing an immuneresponse against a chemokine receptor molecule in a patient. The methodscomprise administering to the patient an immunologically effectiveamount of a pharmaceutical composition comprising an adjuvant and animmunogenic chemokine receptor polypeptide from a extracellular regionof a chemokine receptor molecule, for example CXCR3.

[0007] The immunogenic peptides are preferably conformationallyconstrained, for example by cyclization. The length of the immunogenicpeptide is not critical to the invention. Typically, the peptideconsists of between about 10 and about 50 residues, more often betweenabout 15 and about 30 residues. Exemplary immunogenic peptides of theinvention include MVLEVSDHQVLNDAEVAALL, ENFSSSYDYGENESDSCCTS,PPCPQDFSLNFDRAFLPA, DAAVQWVFGSGLCKV, SAHHDERLNATHCQYN,FPQVGRTALRVLQLVAG, and DILMDLGALARNCGRESRVDVAKS.

[0008] The immunogenic peptides can be administered by any of a numberof means. Typically they are administered parenterally. The adjuvant canbe, for example, alum.

[0009] In preferred embodiments, the method are used to inhibitrecruitment of T cells to inflammation sites in a patient. Typically theinflammatory response is associated with an autoimmune disease, such asmultiple sclerosis.

[0010] The invention also provides pharmaceutical compositions suitablefor use in the above methods.

Definitions

[0011] The term “peptide” is used interchangeably with “oligopeptide” or“polypeptide” in the present specification to designate a series ofresidues, typically L-amino acids, connected one to the other typicallyby peptide bonds between the α-amino and carbonyl groups of adjacentamino acids.

[0012] The term “cyclic peptide” refers to peptides in which theN-terminal residue is linked to the C-terminal residue either directlyor through an intermediate. Examples of links between the two residuesinclude disulfide bonds and thioether linkages as described below.

[0013] An “immunogenic chemokine receptor polypeptide” of the presentinvention is a polypeptide capable of eliciting an immune responseagainst a chemokine receptor molecule associated with inflammation inautoimmune responses in a patient, such as multiple sclerosis. As setforth in more detail below, the sequence of residues in the polypeptidewill be identical to or substantially identical to a polypeptidesequence in a chemokine receptor molecule. Thus, a polypeptide of theinvention that has a sequence “from a extracellular region of achemokine receptor molecule” is polypeptide that has a sequence eitheridentical to or substantially identical to the naturally occurringchemokine receptor amino acid sequence of the region.

[0014] As used herein a “extracellular region” of a chemokine receptormolecule is a region of the molecule which is exposed on the surface ofa cell expressing the native molecule. FIG. 1 provides a schematic ofthe extracellular domains of the human CXCR3 molecule. This molecule hasfour extracellular regions designated as SP-1, SP-2, SP-3 and SP-4,starting from the N-terminus.

[0015] As used herein, the term “adjuvant” refers to any substancewhich, when administered with or before an antigen, increases and/orqualitatively affects the immune response against the antigen in termsof antibody formation and/or the cell-mediated response. Exemplaryadjuvants for use in the present invention are provided below.

[0016] The phrases “isolated” or “biologically pure” refer to materialwhich is substantially or essentially free from components whichnormally accompany it as found in its native state. Thus, the chemokinereceptor polypeptides of this invention do not contain materialsnormally associated with their in situ environment, e.g., other surfaceproteins on T cells. Even where a protein has been isolated to ahomogenous or dominant band, there are trace contaminants in the rangeof 5-10% of native protein which co-purify with the desired protein.Isolated polypeptides of this invention do not contain such endogenousco-purified protein.

[0017] The term “residue” refers to an amino acid or amino acid mimeticincorporated in a oligopeptide by an amide bond or amide bond mimetic.

BRIEF DESCRIPTION OF THE DRAWINGS

[0018]FIG. 1 shows acidification rate changes due to the binding ofanti-human CXCR3 Mab or P-10 to CXCR3-NSO transfectants. 5X10⁵ CXCR3-NSOtransfectant cells and untransfected “BONZO-NSO” cells (negativecontrol) were spotted in the microphysiometer chambers along withagarose and 1, 5, 10 mglml anti-CXCR3 (FIG 1 a) or IP-10 (FIG 1 b) waspumped for 10 min. There is a dose dependent increase in theacidification rates with anti-CXCR3 in NSO-CXCR3 transfectant cells.Untransfected BONZO-NSO cells did not show any change in acidificationrate changes either with the anti-CXCR3 antibody, or IP-10. The arrowindicates the time that ligands were added to the cells.

[0019]FIG. 2 shows the structure of the seven transmembrane G-proteincoupled human CXCR3. Only amino acids in the extracellular domains aregiven

[0020]FIG. 3 is a schematic of the experiments in which peptidesvaccines of the invention were used to prevent EAE in mice.

[0021]FIG. 3 shows the results of experiments in which peptides vaccinesof the invention were used to prevent EAE in mice.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0022] The present invention provides immunogenic polypeptides derivedfrom chemokine receptor protein sequences for use in compositions andmethods for the treatment and prevention of inflammatory responses. Thepolypeptides are capable of inducing an immune response againstchemokine receptor molecules which mediate inflammatory responsesassociated with various diseases. In preferred embodiments thepolypeptides of the invention are conformationally constrained (e.g.,cyclized) and derived from extracellular regions of human CXCR3.Immunization with the polypeptides of the invention provides a specificimmune response to particular chemokine receptors and results in thespecific inhibition of the inflammatory responses mediated by thesemolecules.

[0023] The methods can be used to treat any inflammatory responsemediated by chemokines. In particular, the methods are useful fortreatment of autoimmune diseases, such as multiple sclerosis. Multiplesclerosis (MS), a human demyelinating disease which afflicts 600,000individuals worldwide, results from damage of the myelin sheath ofoligodendroglial cells in the Central Nervous System (CNS). Although thepathogenesis and etiology of MS have not yet been established, it iswidely believed that the disease has an immunological basis and thatboth genetic and environmental factors make a contribution to thepathogenesis. The central mediator of autoimmune attack is believed tobe host CD4+T cells specific for one or more autoantigens in the CNS,with subsequent production of an array of tissue-destructiveinflammatory mediators following autoantigen-activation of these cells.Indeed, immunohistochemical analysis of the focal plaques ofdemyelination which occur in the brains of MS patients as a consequenceof MS pathology have revealed the presence of CD4+T cells infiltratingthese plaques.

[0024] Improved understanding of the immunopathological mechanismsunderlying MS has developed from the study of experimental models ofdemyelination. The most commonly used model, experimental allergicencephalomyelitis (EAE), is an autoimmune inflammatory disorder ofgenetically susceptible mice that is mediated by autoantigen-specificCD4+MHC class II restricted T cells. In susceptible SJL/J mice, thedisease can display a relapsing-remitting clinical course of paralysis,which makes it an ideal system to study the efficacy of variousimmunoregulatory strategies both in the prevention and treatment ofdisease.

[0025] The current invention is focused not on the cytokine-producingphenotype of autoreactive T cells in this disease setting, but on theirtrafficking from the host circulation to the site of pathology, forexample within the CNS in the case of MS. As noted above, hemopoieticcell migration is regulated by chemokines.

[0026] In some embodiments, the invention provides peptide vaccinesagainst the 7-transmembrane chemokine receptor designated CXCR3.Consistent with the chemotactic fingerprint of IP-10, CXCR3 is expressedexclusively on activated effector T lymphocytes. Exemplary peptides ofthe invention include peptides derived from the extracellular domains ofthe CXCR3 protein are presented in Table 1.

[0027] In preferred embodiments, the peptides of the invention arecyclized. Methods for cyclizing peptides are described in detail below.In those cases in which the peptides are cyclized by disulfide linkages,one of skill will recognize that the peptides will further comprisecysteine residues either within the peptide or at each terminus.

[0028] Polypeptides suitable for use in the present invention can beobtained in a variety of ways. Conveniently, they can be synthesized byconventional techniques employing automatic synthesizers, such as theBeckman, Applied Biosystems, or other commonly available peptidesynthesizers using well known protocols. They can also be synthesizedmanually using techniques well known in the art. See, e.g. Stewart andYoung, Solid Phase Peptide Synthesis, (Rockford, Ill., Pierce), 2d Ed.(1984).

[0029] Alternatively, DNA sequences which encode the particularchemokine receptor polypeptide may be cloned and expressed to providethe peptide. Nucleic acid molecules encoding chemokine receptors areknown in the art and sequences of such genes are available, forinstance, from GenBank (see, e.g., GenBank Accession Nos. HSU83326HSU97123, and AF005058).

[0030] Standard techniques can be used to screen cDNA libraries toidentify sequences encoding the desired sequences (see, Sambrook et al.,Molecular Cloning—A Laboratory Manual, Cold Spring Harbor Laboratory,Cold Spring Harbor, New York, 1989). Fusion proteins (those consistingof all or part of the amino acid sequences of two or more proteins) canbe recombinantly produced. In addition, using in vitro mutagenesistechniques, unrelated proteins can be mutated to comprise theappropriate sequences.

[0031] Chemokine receptor proteins from a variety of natural sources arealso conveniently isolated using standard protein purificationtechniques. Peptides can be purified by any of a variety of knowntechniques, including, for example, reverse phase high-performanceliquid chromatography (HPLC), ion-exchange or immunoaffinitychromatography, separation by size, or electrophoresis (See, generally,Scopes, R., Protein Purification, Springer-Verlag, N.Y. (1982)).

[0032] It will be understood that the immunogenic chemokine receptorpolypeptides of the present invention may be modified to provide avariety of desired attributes, e.g., improved pharmacologicalcharacteristics, while increasing or at least retaining substantiallyall of the biological activity of the unmodified peptide. For instance,the peptides can be modified by extending, decreasing the amino acidsequence of the peptide. Substitutions with different amino acids oramino acid mimetics can also be made.

[0033] The peptides employed in the subject invention need not beidentical to peptides disclosed in the Example section, below, so longas the subject peptides are able to induce an immune response againstthe desired chemokine receptor molecule. Thus, one of skill willrecognize that a number of conservative substitutions (described in moredetail below) can be made without substantially affecting the activityof the peptide.

[0034] Single amino acid substitutions, deletions, or insertions can beused to determine which residues are relatively insensitive tomodification. Substitutions are preferably made with small, relativelyneutral moieties such as Ala, Gly, Pro, or similar residues. The effectof single amino acid substitutions may also be probed using D-aminoacids. The number and types of residues which are substituted or addeddepend on the spacing necessary between essential contact points andcertain functional attributes which are sought (e.g., hydrophobicityversus hydrophilicity). Increased immunogenicity may also be achieved bysuch substitutions, compared to the parent peptide. In any event, suchsubstitutions should employ amino acid residues or other molecularfragments chosen to avoid, for example, steric and charge interferencewhich might disrupt binding.

[0035] The substituting amino acids, however, need not be limited tothose naturally occurring in proteins, such as L-α-amino acids, or theirD-isomers. The peptides may be substituted with a variety of moietiessuch as amino acid mimetics well known to those of skill in the art.

[0036] The individual residues of the immunogenic chemokine receptorpolypeptides can be incorporated in the peptide by a peptide bond orpeptide bond mimetic. A peptide bond mimetic of the invention includespeptide backbone modifications well known to those skilled in the art.Such modifications include modifications of the amide nitrogen, theα-carbon, amide carbonyl, complete replacement of the amide bond,extensions, deletions or backbone crosslinks. See, generally, Spatola,Chemistry and Biochemistry of Amino Acids, Peptides and Proteins, Vol.VII (Weinstein ed., 1983). Several peptide backbone modifications areknown, these include, ψ[CH₂S], ψ[CH₂NH], ψ[CSNH₂], ψ[NHCO], ψ[COCH₂] andψ(E) or (Z) CH=CH]. The nomenclature used above, follows that suggestedby Spatola, above. In this context, ψ indicates the absence of an amidebond. The structure that replaces the amide group is specified withinthe brackets.

[0037] Amino acid mimetics may also be incorporated in the peptides. An“amino acid mimetic” as used here is a moiety other than a naturallyoccurring amino acid that conformationally and functionally serves as asubstitute for an amino acid in a polypeptide of the present invention.Such a moiety serves as a substitute for an amino acid residue if itdoes not interfere with the ability of the peptide to illicit an immuneresponse against the appropriate chemokine receptor molecule. Amino acidmimetics may include non-protein amino acids, such as β-γ-δ-amino acids,β-γ-δ-imino acids (such as piperidine-4-carboxylic acid) as well as manyderivatives of L-α-amino acids. A number of suitable amino acid mimeticsare known to the skilled artisan, they include cyclohexylalanine,3-cyclohexylpropionic acid, L-adamantyl alanine, adamantylacetic acidand the like. Peptide mimetics suitable for peptides of the presentinvention are discussed by Morgan and Gainor, (1989) Ann. Repts. Med.Chem. 24:243-252/.

[0038] As noted above, the peptides employed in the subject inventionneed not be identical, but may be substantially identical, to thecorresponding sequence of the target chemokine receptor molecule.Therefore, the peptides may be subject to various changes, such asinsertions, deletions, and substitutions, either conservative ornon-conservative, where such changes might provide for certainadvantages in their use. The polypeptides of the invention can bemodified in a number of ways so long as they comprise a sequencesubstantially identical (as defined below) to a sequence in the targetregion of the chemokine receptor molecule.

[0039] Alignment and comparison of relatively short amino acid sequences(less than about 30 residues) is typically straightforward. Comparisonof longer sequences may require more sophisticated methods to achieveoptimal alignment of two sequences. Optimal alignment of sequences foraligning a comparison window may be conducted by the local homologyalgorithm of Smith and Waterman (1981) Adv. Appl. Math. 2:482, by thehomology alignment algorithm of Needleman and Wunsch (1970) J. Mol.Biol. 48:443, by the search for similarity method of Pearson and Lipman(1988) Proc. Natl. Acad. Sci. (USA) 85:2444, by computerizedimplementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA inthe Wisconsin Genetics Software Package Release 7.0, Genetics ComputerGroup, 575 Science Dr., Madison, Wis.), or by inspection, and the bestalignment (i.e., resulting in the highest percentage of sequencesimilarity over the comparison window) generated by the various methodsis selected.

[0040] The term “sequence identity” means that two polynucleotidesequences are identical (i.e., on a nucleotide-by-nucleotide basis) overa window of comparison. The term “percentage of sequence identity” iscalculated by comparing two optimally aligned sequences over the windowof comparison, determining the number of positions at which theidentical residues occurs in both sequences to yield the number ofmatched positions, dividing the number of matched positions by the totalnumber of positions in the window of comparison (i.e., the window size),and multiplying the result by 100 to yield the percentage of sequenceidentity.

[0041] As applied to polypeptides, the term “substantial identity” meansthat two peptide sequences, when optimally aligned, such as by theprograms GAP or BESTFIT using default gap weights, share at least 80percent sequence identity, preferably at least 90 percent sequenceidentity, more preferably at least 95 percent sequence identity or more(e.g., 99 percent sequence identity). Preferably, residue positionswhich are not identical differ by conservative amino acid substitutions.Conservative amino acid substitutions refer to the interchangeability ofresidues having similar side chains. For example, a group of amino acidshaving aliphatic side chains is glycine, alanine, valine, leucine, andisoleucine; a group of amino acids having aliphatic-hydroxyl side chainsis serine and threonine; a group of amino acids having amide-containingside chains is asparagine and glutamine; a group of amino acids havingaromatic side chains is phenylalanine, tyrosine, and tryptophan; a groupof amino acids having basic side chains is lysine, arginine, andhistidine; and a group of amino acids having sulfur-containing sidechains is cysteine and methionine. Preferred conservative amino acidssubstitution groups are: valine-leucine-isoleucine,phenylalanine-tyrosine, lysine-arginine, alanine-valine, andasparagine-glutamine.

[0042] The polypeptides of the invention typically comprise at leastabout 10 residues and more preferably at least about 15 residues fromthe extracellular domain of a chemokine receptor. In certain embodimentsthe peptides will not exceed about 50 residues and typically will notexceed about 30 residues. For instance, the peptides described belowconsist of about 15 to about 25 residues.

[0043] In the preferred embodiments of the invention, the immunogenicpeptides are conformationally constrained. Means for achieving this arewell known in the art (see, e.g., Hruby and Bonner in Methods inMolecular Biology, Volume 35: Peptide Synthesis Protocols Pennington andDunn eds (Humana Press, Totowa, N.J., 1994). A preferred means forpreparing conformationally constrained peptides is through cyclization.Any method commonly used to produce cyclized oligopeptides can be usedto produce the peptides of the invention. For example, in certainembodiments the peptides will include cysteine residues at both termini,which allow the production of cyclic peptides through disulfidelinkages. Treatment of a such a peptide with an oxidizing agent such asoxygen, iodine or similar agent will produce a cyclic peptide which maybe further purified using chromatographic or other methods of chemicalpurification. Construction of cyclic peptides can also be accomplishedthrough thioether linkages. For instance, N-bromoacetyl-derivatizedpeptides can be reacted with sulfhydryl-containing residues, such ascysteine. Cyclization occurs by reaction of the free sulfhydryl ofcysteine in the peptide with the bromoacetyl group to form a thioetherlinkage (Robey et al., Anal. Biochem. 177:373-7 (1989) and U.S. Pat. No.5,066,716).

[0044] Other methods of constructing cyclic peptides are known to thoseskilled in the art. These include side chain-side chain, side chain-mainchain and main chain-main chain cyclizations. In addition, linkers canbe used to join the amino and carboxyl termini of a peptide. The linkeris capable of forming covalent bonds to both the amino and carboxylterminus. Suitable linkers are well known to those of skill in the artand include, but are not limited to, straight or branched-chain carbonlinkers, heterocyclic carbon linkers, or peptide linkers. The linkersmay be joined to the carboxyl and amino terminal amino acids throughtheir side groups (e.g., through a disulfide linkage to cysteine) orthrough the alpha carbon amino and carboxyl groups of the terminal aminoacids.

[0045] For a general discussion of suitable methods for cyclization see,Hruby and Bonner in Methods in Molecular Biology, Volume 35: PeptideSynthesis Protocols Pennington and Dunn eds (Humana Press, Totowa, N.J.,1994). For instance, cyclizations may include formation of carba analogsand thioethers (Lebl et al. in Peptides 1986 Proceedings of the 19thEuropean Peptide Symposium pp. 341-344; Robey et al., Anal Biochem.177:373-7 (1989) and U.S. Pat. No. 5,066,716), bis-thioethers (Mosberget al. JACS 107:2986-2987 (1985)), azopeptides (Siemion et al. Mol.Cell. Biochem. 34: (1991)), and other cyclic structures, such asbridging structures (Charpentier, M., et al., J Med. Chem.32(6):1184-1190 (1989), Thaisrivongs, S., et al., J. Med. Chem.34(4):127 (1991) and Ozeki, E., et al., Int. J. Peptide Protein Res.34:111 (1989)). Cyclization from backbone-to-backbone positions may alsobe used.

[0046] Bridging is a special type of cyclization in which distant sitesin a peptide are brought together using separate bridging molecules orfragments. Bridging molecules may include, for example, succinicanhydride molecules (Charpentier, B., et al., supra), andcarboxymethylene fragments (Thaisrivongs, S., et al., supra). Bridgingby metals can also be used (Ozeki, E., et al., supra).

[0047] In some embodiments, the peptides include two or more cystineresidues. The cystines can be substituted or added within the peptide orat either terminus. The position of the cystines is not critical so longas disulfide linkages can form between them which allow the productionof cyclic peptides. For example, treatment of such a peptide with anoxidizing agent such as oxygen, iodine or similar agent will produce acyclic peptide which may be further purified using chromatographic orother methods of chemical purification.

[0048] In addition to use of peptides, antibodies raised againstpeptides of the invention can be used to inhibit inflammatory responses.Antibodies can be raised to the peptides of the present invention usingtechniques well known to those of skill in the art. Anti-idiotypicantibodies can also be generated. The following discussion is presentedas a general overview of the techniques available; however, one of skillwill recognize that many variations upon the following methods areknown.

[0049] A number of immunogens can be used to produce antibodiesspecifically reactive with the peptides. For instance, the entirechemokine receptor molecule or fragments containing the desired sequencecan be used. Synthetic peptides as disclosed here can be used either inlinear form or cyclized.

[0050] Methods of producing polyclonal antibodies are known to those ofskill in the art. In brief, an immunogen (antigen), preferably apurified polypeptide, a polypeptide coupled to an appropriate carrier(e.g., GST, keyhole limpet hemanocyanin, etc.), or a polypeptideincorporated into an immunization vector such as a recombinant vaccimiavirus (see, U.S. Pat. No. 4,722,848) is mixed with an adjuvant andanimals are immunized with the mixture. The animal's immune response tothe immunogen preparation is monitored by taking test bleeds anddetermining the titer of reactivity to the polypeptide of interest. Whenappropriately high titers of antibody to the immunogen are obtained,blood is collected from the animal and antisera are prepared. Furtherfractionation of the antisera to enrich for antibodies reactive to thepolypeptide is performed where desired (see, e.g., Coligan (1991)Current Protocols in Immunology Wiley/Greene, N.Y.; and Harlow and Lane(1989) Antibodies: A Laboratory Manual Cold Spring Harbor Press, N.Y.).

[0051] In some instances, it is desirable to prepare monoclonalantibodies from various mammalian hosts, such as mice, rodents,primates, humans, etc. Description of techniques for preparing suchmonoclonal antibodies are found in, e.g., Stites et al. (eds.) Basic andClinical Immunology (4th ed.) Lange Medical Publications, Los Altos,Calif., and references cited therein; Harlow and Lane, Supra; Goding(1986) Monoclonal Antibodies: Principles and Practice (2d ed.) AcademicPress, New York, N.Y.; and Kohler and Milstein (1975) Nature256:495-497. Summarized briefly, this method proceeds by injecting ananimal with an immunogen. The animal is then sacrificed and cells takenfrom its spleen, which are fused with myeloma cells. The result is ahybrid cell or “hybridoma” that is capable of reproducing in vitro. Thepopulation of hybridomas is then screened to isolate individual clones,each of which secrete a single antibody species to the immunogen. Inthis manner, the individual antibody species obtained are the productsof immortalized and cloned single B cells from the immune animalgenerated in response to a specific site recognized on the immunogenicsubstance.

[0052] Alternative methods of immortalization include transformationwith Epstein Barr Virus, oncogenes, or retroviruses, or other methodsknown in the art. Colonies arising from single immortalized cells arescreened for production of antibodies of the desired specificity andaffinity for the antigen, and yield of the monoclonal antibodiesproduced by such cells is enhanced by various techniques, includinginjection into the peritoneal cavity of a vertebrate (preferablymammalian) host. Specific monoclonal and polyclonal antibodies willusually bind with a K_(D) of at least about 0.1 mM, more usually atleast about 50 μM, and most preferably at least about 1 μM or better.

[0053] Other suitable techniques involve selection of libraries ofrecombinant antibodies in phage or similar vectors (see, e.g., Huse etal. (1989) Science 246:1275-1281; and Ward, et al. (1989) Nature341:544-546; and Vaughan et al (1996) Nature Biotechnology, 14:309-314).

[0054] Frequently, the peptides and antibodies of the invention will belabeled by joining, either covalently or non-covalently, a substancewhich provides for a detectable signal. A wide variety of labels andconjugation techniques are known and are reported extensively in boththe scientific and patent literature. Suitable labels includeradionucleotides, enzymes, substrates, cofactors, inhibitors,fluorescent moieties, chemiluminescent moieties, magnetic particles, andthe like. Patents teaching the use of such labels include U.S. Pat. Nos.3,817,837; 3,850,752; 3,939,350; 3,996,345; 4,277,437; 4,275,149; and4,366,241. Also, recombinant immunoglobulins may be produced. See,Cabilly, U.S. Pat. No. 4,816,567; and Queen et al. (1989) Proc. Nat'lAcad. Sci. USA 86: 10029-10033.

[0055] The antibodies of this invention can also be administered to anorganism (e.g., a human patient) for therapeutic purposes (e.g., toinhibit an autoimmune response). Antibodies administered to an organismother than the species in which they are raised are often immunogenic.Thus, for example, murine antibodies administered to a human ofteninduce an immunologic response against the antibody (e.g., the humananti-mouse antibody (HAMA) response) on multiple administrations. Theimmunogenic properties of the antibody are reduced by altering portions,or all, of the antibody into characteristically human sequences therebyproducing chimeric or human antibodies, respectively.

[0056] Chimeric antibodies are immunoglobulin molecules comprising ahuman and non-human portion. More specifically, the antigen combiningregion (or variable region) of a humanized chimeric antibody is derivedfrom a non-human source (e.g., murine) and the constant region of thechimeric antibody (which confers biological effector function to theimmunoglobulin) is derived from a human source. The chimeric antibodyshould have the antigen binding specificity of the non-human antibodymolecule and the effector function conferred by the human antibodymolecule. A large number of methods of generating chimeric antibodiesare well known to those of skill in the art (see, e.g., U.S. Pat. Nos:5,502,167, 5,500,362, 5,491,088, 5,482,856, 5,472,693, 5,354,847,5,292,867, 5,231,026, 5,204,244, 5,202,238, 5,169,939, 5,081,235,5,075,431, and 4,975,369). An alternative approach is the generation ofhumanized antibodies by linking the CDR regions of non-human antibodiesto human constant regions by recombinant DNA techniques. See Queen etal., Proc. Natl. Acad. Sci. USA 86:10029-10033 (1989) and WO 90/07861.

[0057] In one preferred embodiment, recombinant DNA vector is used totransfect a cell line that produces an antibody against a peptide of theinvention. The novel recombinant DNA vector contains a “replacementgene” to replace all or a portion of the gene encoding theimmunoglobulin constant region in the cell line (e.g., a replacementgene may encode all or a portion of a constant region of a humanimmunoglobulin, or a specific immunoglobulin class), and a “targetsequence” which allows for targeted homologous recombination withimmunoglobulin sequences within the antibody producing cell.

[0058] In another embodiment, a recombinant DNA vector is used totransfect a cell line that produces an antibody having a desiredeffector function, (e.g., a constant region of a human immunoglobulin)in which case, the replacement gene contained in the recombinant vectormay encode all or a portion of a region of an antibody and the targetsequence contained in the recombinant vector allows for homologousrecombination and targeted gene modification within the antibodyproducing cell. In either embodiment, when only a portion of thevariable or constant region is replaced, the resulting chimeric antibodymay define the same antigen and/or have the same effector function yetbe altered or improved so that the chimeric antibody may demonstrate agreater antigen specificity, greater affinity binding constant,increased effector function, or increased secretion and production bythe transfected antibody producing cell line, etc.

[0059] In another embodiment, this invention provides for fully humanantibodies. Human antibodies consist entirely of characteristicallyhuman polypeptide sequences. The human antibodies of this invention canbe produced in using a wide variety of methods (see, e.g., Larrick etal., U.S. Pat. No. 5,001,065). In one preferred embodiment, the humanantibodies of the present invention are produced initially in triomacells. Genes encoding the antibodies are then cloned and expressed inother cells, particularly, nonhuman mammalian cells. The generalapproach for producing human antibodies by trioma technology has beendescribed by Ostberg et al. (1983), Hybridoma 2:361-367, Ostberg, U.S.Pat. No. 4,634,664, and Engelman et al., U.S. Pat. No. 4,634,666. Theantibody-producing cell lines obtained by this method are called triomasbecause they are descended from three cells; two human and one mouse.Triomas have been found to produce antibody more stably than ordinaryhybridomas made from human cells.

[0060] Formulation and Administration

[0061] The peptides or antibodies (typically monoclonal antibodies) ofthe present invention and pharmaceutical compositions thereof are usefulfor administration to mammals, particularly humans, to treat and/orprevent deleterious immune inflammatory responses, particularly thoseassociated with autoimmune responses. Over 30 autoimmune diseases arepresently known, including myasthenia gravis (MG), multiple sclerosis(MS), systemic lupus erythematosis (SLE), rheumatoid arthritis (RA),insulin-dependent diabetes mellitus (IDDM), and the like. Suitableformulations are found in Remington's Pharmaceutical Sciences, MackPublishing Company, Philadelphia, PA, 17th ed. (1985).

[0062] The immunogenic peptides or antibodies of the invention areadministered prophylactically or to an individual already suffering fromthe disease. The peptide compositions are administered to a patient inan amount sufficient to elicit an effective immune response to thechemokine receptor molecule from which the peptides are derived. Aneffective immune response is one that inhibits recruitment of T cells tosites of inflammation. An amount adequate to accomplish this is definedas “therapeutically effective dose” or “immunogenically effective dose.”Amounts effective for this use will depend on, e.g., the peptidecomposition, the manner of administration, the stage and severity of thedisease being treated, the weight and general state of health of thepatient, and the judgment of the prescribing physician, but generallyrange for the initial immunization (that is for therapeutic orprophylactic administration) from about 0.1 mg to about 1.0 mg per 70kilogram patient, more commonly from about 0.5 mg to about 0.75 mg per70 kg of body weight. Boosting dosages are typically from about 0.1 mgto about 0.5 mg of peptide using a boosting regimen over weeks to monthsdepending upon the patient's response and condition. A suitable protocolwould include injection at time 0, 4, 2, 6, 10 and 14 weeks, followed byfurther booster injections at 24 and 28 weeks.

[0063] It must be kept in mind that the peptides and compositions of thepresent invention may generally be employed in serious disease states,that is, life-threatening or potentially life threatening situations. Insuch cases, in view of the minimization of extraneous substances and therelative nontoxic nature of the peptides, it is possible and may be feltdesirable by the treating physician to administer substantial excessesof these peptide compositions.

[0064] For therapeutic use, administration should begin at the firstsign of autoimmune disease. This is followed by boosting doses until atleast symptoms are substantially abated and for a period thereafter. Insome circumstances, loading doses followed by boosting doses may berequired. The resulting immune response helps to cure or at leastpartially arrest symptoms and/or complications. Vaccine compositionscontaining the peptides are administered prophylactically to a patientsusceptible to or otherwise at risk of the disease to elicit an immuneresponse against the target Chemokine receptor antigen.

[0065] The pharmaceutical compositions (containing either peptides orantibodies) are intended for parenteral or oral administration.Preferably, the pharmaceutical compositions are administeredparenterally, e.g., subcutaneously, intradernally, or intramuscularly.Thus, the invention provides compositions for parenteral administrationwhich comprise a solution of the immunogenic peptides dissolved orsuspended in an acceptable carrier, preferably an aqueous carrier. Avariety of aqueous carriers may be used, e.g., water, buffered water,0.4% saline, 0.3% glycine, hyaluronic acid and the like. Thesecompositions may be sterilized by conventional, well known sterilizationtechniques, or may be sterile filtered. The resulting aqueous solutionsmay be packaged for use as is, or lyophilized, the lyophilizedpreparation being combined with a sterile solution prior toadministration. The compositions may contain pharmaceutically acceptableauxiliary substances as required to approximate physiologicalconditions, such as buffering agents, tonicity adjusting agents, wettingagents and the like, for example, sodium acetate, sodium lactate, sodiumchloride, potassium chloride, calcium chloride, sorbitan monolaurate,triethanolamine oleate, etc.

[0066] For solid compositions, conventional nontoxic solid carriers maybe used which include, for example, pharmaceutical grades of mannitol,lactose, starch, magnesium stearate, sodium saccharin, talcum,cellulose, glucose, sucrose, magnesium carbonate, and the like. For oraladministration, a pharmaceutically acceptable nontoxic composition isformed by incorporating any of the normally employed excipients, such asthose carriers previously listed, and generally 10-95% of activeingredient, that is, one or more peptides of the invention, and morepreferably at a concentration of 25%-75%.

[0067] As noted above, the peptide compositions are intended to inducean immune response to the peptides. Thus, compositions and methods ofadministration suitable for maximizing the immune response arepreferred. For instance, peptides may be introduced into a host,including humans, linked to a carrier or as a homopolymer orheteropolymer of active peptide units. Alternatively, the a “cocktail”of polypeptides can be used. A mixture of more than one polypeptide hasthe advantage of increased immunological reaction and, where differentpeptides are used to make up the polymer, the additional ability toinduce antibodies to a number of epitopes. For instance, polypeptidescomprising sequences from extracellular regions of α and β chains may beused in combination. Useful carriers are well known in the art, andinclude, e.g., KLH, thyroglobulin, albumins such as human serum albumin,tetanus toxoid, polyamino acids such as poly(lysine:glutamic acid),influenza, hepatitis B virus core protein, hepatitis B virus recombinantvaccine and the like.

[0068] The use of more than one polypeptide is particularly useful toenhance the immune response against polypeptides of the invention. Asdemonstrated below, although the polypeptides may be derived from selfChemokine receptor molecules expressed in the patient, they can inducean immune response. In some instances, the immune response to the selfpolypeptide may not be sufficiently strong. In these instances, it maybe necessary to break tolerance to the polypeptide. The compositions maycomprise one or more of the foreign polypeptides that are sufficientlysimilar to the self polypeptides to induce an immune response againstboth the foreign and self polypeptides (see, Mamula et al. J. Immunol.149:789-795 (1992). Suitable proteins include synthetic polypeptidesdesigned for this purpose or polypeptide sequences from homologousproteins from natural sources, such as proteins encoded by a differentallele at the same locus as the self polypeptide.

[0069] The compositions also include an adjuvant. As used here, numberof adjuvants are well known to one skilled in the art. Suitableadjuvants include incomplete Freund's adjuvant, alum, aluminumphosphate, aluminum hydroxide,N-acetyl-muramyl-L-threonyl-D-isoglutamine (thr-MDP),N-acetyl-nor-muramyl-L-alanyl-D-isoglutamine (CGP 11637, referred to asnor-MDP),N-acetylmuramyl-Lalanyl-D-isoglutaminyl-L-alanine-2-(1′-2′-dipalmitoyl-sn-glycero-3-hydroxyphosphoryloxy)-ethylamine(CGP 19835A, referred to as MTP-PE), and RIBI, which contains threecomponents extracted from bacteria, monophosphoryl lipid A, trehalosedimycolate and cell wall skeleton (NPL+TDM+CWS) in a 2% squalene/Tween80 emulsion. The effectiveness of an adjuvant may be determined bymeasuring the amount of antibodies directed against the immunogenicpeptide.

[0070] A particularly useful adjuvant and immunization schedule aredescribed in Kwak et al. New Eng. J Med. 327-1209-1215 (1992). Theimmunological adjuvant described there comprises 5% (wt/vol) squalene,2.5% Pluronic L121 polymer and 0.2 % polysorbate in phosphate bufferedsaline.

[0071] The concentration of immunogenic peptides of the invention in thepharmaceutical formulations can vary widely, i.e. from less than about0.1%, usually at or at least about 2% to as much as 20% to 50% or moreby weight, and will be selected primarily by fluid volumes, viscosities,etc., in accordance with the particular mode of administration selected.

[0072] The peptides of the invention can also be expressed by attenuatedviral hosts, such as vaccinia or fowlpox. This approach involves the useof vaccinia virus as a vector to express nucleotide sequences thatencode the peptides of the invention. Upon introduction into a host, therecombinant vaccinia virus expresses the immunogenic peptide, andthereby elicits an immune response. Vaccinia vectors and methods usefulin immunization protocols are described in, e.g., U.S. Pat. No.4,722,848. Another vector is BCG (Bacille Calnette Guerin). BCG vectorsare described in Stover et al. (Nature 351:456-460 (1991)). A widevariety of other vectors useful for therapeutic administration orimmunization of the peptides of the invention, e.g., Salmonella typhivectors and the like, will be apparent to those skilled in the art fromthe description herein.

[0073] The DNA encoding one or more of the peptides of the invention canalso be administered to the patient. This approach is described, forinstance, in Wolff et. al., Science 247:1465-1468 (1990) as well as U.S.Pat. Nos. 5,580,859 and 5,589,466.

[0074] In order to enhance serum half-life, the peptides may also beencapsulated, introduced into the lumen of liposomes, prepared as acolloid, or other conventional techniques may be employed which providean extended serum half-life of the peptides. A variety of methods areavailable for preparing liposomes, as described in, e.g., Szoka et al.,Ann. Rev. Biophys. Bioeng. 9:467 (1980), U.S. Pat. Nos. 4,235,871,4,501,728 and 4,837,028.

[0075] The peptides or antibodies of the invention can also be used fordiagnostic purposes. For instance, peptides can be used to screen forautoantibodies to ensure that the vaccination has been effective.Antibodies can be used to detect the presence of particular Chemokinereceptor molecules associated with disease.

[0076] The following examples are offered by way of illustration, not byway of limitation.

EXAMPLE 1

[0077] This example describes a reproducible bioassay for CXCR3activation.

[0078] A stable line transfectant of NSO-1 cells expressing the cDNA forhuman CXCR3 was prepared according to standard techniques. The surfaceexpression of hu-CXCR3 on the transfectants compared to untransfectedparent NSO-1 cells was confirmed by FACS staining using mouse anti-humanCXCR3 monoclonal antibody (R&D systems).

[0079] To establish a bioassay for CXCR3 activation, the CXCR3transfectant cells were cultured with either human IP-10, or the mouseanti-human CXCR3 antibody, and the physiological response of the cellswas measured using a microphysiometer. This machine measures changes inthe pH of the extracellular medium of the cell cultures which resultfrom ligand receptor binding on the cell surface. These extracellularacidification rate measurements have previously been used as markers ofantigen specific T cell activation and T cell epitope identification.The current assay is used to provide a biological read out for theidentification of chemokine or chemokine receptor peptides involved inbinding to IP- 10 or agonistic anti-CXCR3 antibody.

[0080] The experiments indicated that human IP-10 and anti-CXCR3antibody both triggered substantial acidification rate changes in theCXCR3 transfectants, as shown in FIG. 1. Importantly, the same ligandsinduce no change in acidification rate of untransfected NSO-1 cells(FIG. 1).

EXAMPLE 2

[0081] This example describes synthesis of human CXCR3 derived peptides.

[0082] A schematic representation of the surface portion of human CXCR3is given in FIG. 2. The receptor has 4 surface portions, 4 intracellularportions and seven transmembrane portions. The surface portions weredesignated starting from N-terminus as SP-1, SP-2, SP-3 and SP-4.

[0083] The peptides shown in Table 1 derived from these four surfaceportions were prepared by solid phase peptide synthesis. The names ofthe peptides are based on the surface portion of CXCR3 from which theywere derived. For example, SP-1-1 means this peptide was derived fromthe first portion of the CXCR3 protein. TABLE 1 Peptide Name SequenceSP-1-1 MVLEVSDHQVLNDAEVAALL-NH2 SP-1-2 ENFSSSYDYGENESDSCCTS-NH2 SP-1-3PPCPQDFSLNFDRAFLPA-NH2 SP-2-1 DAAVQWVFGSGLCKV-NH2 SP-3-1SAHHDERLNATHCQYN-NH2 SP-3-2 FPQVGRTALRVLQLVAG-NH2 SP-4-1DILMDLGALARNCGRESRVDVAKS-NH2

[0084] The ability of these receptor-derived peptides to bind anti-CXCR3antibody was evaluated using a standard ELISA format. The peptides weredissolved in 0.1 M bicarbonate buffer and coated on a 96 well ELISAplate overnight. The excess peptides were removed and nonspecificbinding sites in the wells were blocked by 0.1 % bovine serum albumin.Anti-CXCR3 antibody (0.5 μg/well) was added to these wells and incubatedfor 2 hours. Excess antibody was removed by washing with PBS. HRPconjugated goat anti-mouse antibody was used as secondary antibody fordetection. Two of the seven receptor-derived peptides, namely SP-1-3 andSP-4-1, showed substantial binding to anti-CXCR3 antibody. Furthersupport for the conclusion that anti-CXCR3 antibody bound two of theseven receptor-derived peptides was provided by FACS analysis of thesame interactions. These studies revealed that the receptor-derivedpeptide SP-4-1 potently blocked binding of anti-CXCR3 antibody to theCXCR3 cell line transfectant. The receptor-derived peptide SP-1-3provided partial inhibition of the binding of the antibody to CXCR3transfectants. In contrast, an antibody nonbinding receptor-derivedpeptide, SP-2-1, failed to inhibit binding to CXCR3 transfectants. Thedata described here collectively demonstrate that anti-CXCR3 antibodycan bind to two separate peptide portions of the CXCR3 extracellulardomain.

[0085] EXAMPLE 3

[0086] This example demonstrates that peptide vaccines of the inventioncan be used to prevent EAE in mice.

[0087] Protocols for animal experiments:

[0088] SJL mice (6-8 weeks old) were obtained from Jackson Laboratories.They were kept in quarantine for two weeks. These mice get EAE whenimmunized with a peptide from proteolipid protein (PLP). The peptidesequence used for the immunization of these mice is PLP 139-151 and isamidated at the C terminus (HSLGWLGHPDKF-NH2). For the experiments,induction of the disease is considered Day 0. Three weeks before theinduction of the disease, the mice were vaccinated with HumanCXCR3S-P4-1 peptide mixed with complete Freund's Adjuvant.

[0089] Preparation of peptide CFA emulsion for treatment

[0090] 4mg of human CXCR3 SP-4-1 peptide was dissolved in 1 ml ofphosphate buffered saline pH 7.4 (PBS). One ml of CFA obtained from VWR(Difco, Adjuvant Complete H37RA) was added to the peptide solution andthe mixture was sonicated for 5 seconds using a fine tip sonicator. Theemulsion was taken in 1 ml syringe (needed 2 syringes) fitted with a 25gauge needle. Each mouse was given 100 μl of the emulsin under eachflank near the hind legs by subcutaneous injection (total volume permouse=200 μl, total peptide per mouse=200 μg).

[0091] Preparation of CFA alone:

[0092] One ml of PBS and 1 ml of CFA were mixed and sonicated for 5seconds and the emulsin is drawn into a 1 ml syringe fitted with a 25gauge needle. Each mouse was given 100 μl of this emulsion under eachflank near hind legs by subcutaneous injection (total volume of CFA permouse=200 μl).

[0093] Induction of Disease:

[0094] To an aqueous solution of peptide (4 mg per ml in PBS) equalvolume of CFA was added and the mixture was sonicated for 5 seconds. Theemulsion was drawn in a 1 ml syringe fitted with a 25 gauge needle andsubcutaneously injected (200 μl total per mouse) in mice at the foot padand back.

[0095] Testing the antibody response to SP4-1 peptide

[0096] The mice were bled at week 0 and week 5 and serum was tested byELISA for the presence of antibodies against the human CXCR3 SP-4-1peptide. Briefly, the SP4-1 peptide dissolved in 0.1 M sodiumbicarbonate buffer was plated on a 96-well ELISA plate overnight. Thenonspecific binding sites on the plate were coated with 0.1% bovineserum albumin solution in PBS. The wells were washed and serum (dilutedin PBS) was added to the wells and incubated at room temperature for 1.5hours. The wells were then washed and HRP conjugated anti-mouseimmunoglobulin antibody was used to detect the presence of anti-SP4-1antibodies). The results of this ELISA clearly indicate that the SP4-1treated mice show an antibody response against this peptide.

[0097] Results of EAE study

[0098] Sp4-1 was administered with CFA at 21 days and 14 days beforeinduction of EAE as described above (FIG. 3). The results are shown inFIG. 4. There it can be seen that while most of the untreated and CFAtreated mice showed clinical symptoms of EAE, 7 out of 8 mice treatedwith SP4-1 in CFA showed no clinical symptoms.

[0099] The above examples are provided to illustrate the invention butnot to limit its scope. Other variants of the invention will be readilyapparent to one of ordinary skill in the art and are encompassed by theappended claims. All publications, patents, and patent applicationscited herein are hereby incorporated by reference.

What is claimed is:
 1. A method of inducing an immune response against achemokine receptor molecule in a patient, the method comprisingadministering to the patient an immunologically effective amount of apharmaceutical composition comprising an adjuvant and an immunogenicchemokine receptor polypeptide from a extracellular region of achemokine receptor molecule.
 2. The method of claim 1 , wherein thechemokine receptor is CXCR3.
 3. The method of claim 1 , wherein theimmunogenic peptide is conformationally constrained.
 4. The method ofclaim 1 , wherein the immunogenic peptide is cyclized.
 5. The method ofclaim 1 , wherein the immunogenic chemokine receptor peptide consists ofbetween about 10 and about 50 residues.
 6. The method of claim 1 ,wherein the immunogenic chemokine receptor peptide consists of betweenabout 15 and about 30 residues.
 7. The method of claim 1 , wherein theimmunogenic chemokine receptor polypeptide has an amino acid sequenceselected from the group consisting of MVLEVSDHQVLNDAEVAALL,ENFSSSYDYGENESDSCCTS, PPCPQDFSLNFDRAFLPA, DAAVQWVFGSGLCKV,SAHHDERLNATHCQYN, FPQVGRTALRVLQLVAG, and DILMDLGALARNCGRESRVDVAKS. 8.The method of claim 1 , wherein the administration is parenteral.
 9. Themethod of claim 1 , wherein the adjuvant is alum.
 10. A method ofinhibiting recruitment of T cells to inflammation site in a patient, themethod comprising administering to the patient an immunologicallyeffective amount of a pharmaceutical composition comprising an adjuvantand an immunogenic chemokine receptor peptide from a extracellularregion of a chemokine receptor molecule.
 11. The method of claim 10 ,wherein the chemokine receptor is CXCR3.
 12. The method of claim 10 ,wherein the immunogenic peptide is conformationally constrained.
 13. Themethod of claim 10 , wherein the immunogenic peptide is cyclized. 14.The method of claim 10 , wherein the immunogenic chemokine receptorpeptide consists of between about 10 and about 50 residues.
 15. Themethod of claim 10 , wherein the immunogenic chemokine receptor peptideconsists of between about 15 and about 30 residues.
 16. The method ofclaim 10 , wherein the immunogenic chemokine receptor polypeptide has anamino acid sequence selected from the group consisting ofMVLEVSDHQVLNDAEVAALL, ENFSSSYDYGENESDSCCTS, PPCPQDFSLNFDRAFLPA,DAAVQWVFGSGLCKV, SAHHEDERLNATHCQYN, FPQVGRTALRVLQLVAG, andDILMDLGALARNCGRESRVDVAKS.
 17. The method of claim 10 , wherein theinflammatory response is associated with multiple sclerosis.
 18. Apharmaceutical composition comprising an adjuvant and an isolatedimmunogenic chemokine receptor polypeptide from a extracellular regionof a chemokine receptor molecule.
 19. The composition of claim 18 ,wherein the chemokine receptor is CXCR3.
 20. The composition of claim 18, wherein the immunogenic peptide is conformationally constrained. 21.The composition of claim 18 , wherein the immunogenic peptide iscyclized.
 22. The composition of claim 18 , wherein the immunogenicchemokine receptor peptide consists of between about 10 and about 50residues.
 23. The composition of claim 18 , wherein the immunogenicchemokine receptor peptide consists of between about 15 and about 30residues.
 24. The composition of claim 18 , wherein the immunogenicchemokine receptor polypeptide has an amino acid sequence selected fromthe group consisting of MVLEVSDHQVLNDAEVAALL, ENFSSSYDYGENESDSCCTS,PPCPQDFSLNFDRAFLPA, DAAVQWVFGSGLCKV, SAHHDERLNATHCQYN,FPQVGRTALRVLQLVAG, and DILMDLGALARNCGRESRVDVAKS.